Performing Department
Food Science & Technology
Non Technical Summary
Although recent estimates suggest that the upward trend of obesity prevalence has plateaued in 2013 the first time in the last 20years, obesity continues to be a leading public health problem in the U.S. Based on the 2012 statistics provided by CDC, noneof the U.S. state has obesity prevalence lower than 20%. In particular, obesity prevalence in Mississippi State is the secondhighest in the U.S. (34.6%). Common bean production in Mississippi has been declined, partially due to the increasedproduction of soybeans over the years. Nonetheless, dry beans are one of the most nutritious foods offering proteins, fiber andbioactive compounds. Considering the relative low economic status in the Southwest and high obesity incidence, offering aninexpensive diet with high nutritious value together with obesity suppression capability is a strategy towards food for healthytargeting the specific geographic area.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
Analyze, document, and utilize genomic resources to enhance nutritional qualities and identify diversity within Phaseolus vulgaris to facilitate development of nutritious food products to promote human health and well-being.
Project Methods
Through laboratory work, completion of this proposal shall advance the field of diet and optimal health by offering science-basedknowledge. Non-digestible dietary fibers from selected dry beans have been extracted and used for fermentation by animal feces thatcontainintestinal bacteria. In the current proposal as detailed below, the fermented products will be applied to mouse models of adipogenesis. Suchresults will be evaluated and interpreted by comparing adipogenesis with or without the dry bean extract products. These results are expected toprovide invaluable information that can impact on food choices of the intended audiences.Diet preparation.Cooked black bean and non-digestible fiber isolation. To prepare black beans for the diets and to simulate what common beans are typically cooked for human consumption, 50 kg of black beans will be soaked overnight in distilled water and then cooked at 98 °C for about 1 h until they are soft based on finger compression test. Cooked beans and broth will be ground in a grinder, lyophilized by using a freeze-drier, pass through a 60 mm mesh size and stored in vacuumed bags at -20 °C. We will follow the AOAC method 991.43 and our previous approach for non-digestible fiber extraction. Briefly, 1 kg of each of the lyophilized bean flour will be placed in Erlenmeyer flasks containing 50 L phosphate buffer (0.08 mol/L, pH 6) and pH adjusted to 6 with 0.375 M HCl or 0.275 M NaOH. Samples will be added with 100 mL of thermo stable α-amylase and placed in a water bath at 100 °C for 30 min with stirring every 5 min. After being cooled down at room temperature, the samples will be adjusted to pH 7.5, added with 100 mL of protease (5 mg/mL in PBS), and incubated at 60 °C for 30 min. After being cooled down, samples will be adjusted to pH 4, placed in a water bath at 60 °C for 30 min, then added and incubated with 300 mL of amyloglucosidase for another 30 min at 60 °C. Then samples are added with 95% ethanol at 1:4 sample:ethanol ratio and left at room temperature for 24 h. Samples are filtered by 3MM paper and washed 3 times by 10 L of distilled water. The residues are placed in an oven at 90 °C for 2 h and then weighted, which is considered non-digestible fiber.Diets. For experiment 1 (wild-type mice), we use AIN-93G as a control (C) diet (3.9 kcal/g), an AIN-93G-based high fat (F) diet (5.5 kcal/g, BioServ Inc., ref 22), and the high fat diet with cellulose being replaced by the non-digestible fiber isolated from black bean as detailed above (FF) or the majority of energy sources replaced by whole black beans (FW). For experiment 2 (ob/ob mice) , we use AIN-93G diet or the diet with cellulose being replaced by non-digestible fiber isolated from black beans (CF) or the majority of energy sources replaced by whole black beans (CW).Mice. C57BL/6J wild-type mice and ob/ob mice on a C57BL/6J background (B6.V-Lepob/J) will be purchased from the Jackson Lab. Mice at 3-4 weeks of age will be fed with the above-described diets for 8 weeks, followed by blood and organ collection. Based on power analyses and published data, n=8 per group is expected to provide adequate power (>95%) to detect a significant difference in assays between the dietary groups in experiments 1 and 2.Assays.Mouse growth and metabolism-related phenotypes. Body weight and feed intake will be assessed weekly. Femurs cleaned with adherent tissues will be analyzed by micro-computated tomography (microCT) to determine trabecular and cortical bone structural indices, including bone volume, connectivity density, cortical thickness, trabecular number, separation and thickness. Except for insulin, leptin and IGF-1, other blood markers (triglycerides, cholesterol, glucose, amylase, Asp aminotransferase, Ala aminotransferase, creatine phosphokinase, lactate dehydrogenase, alkaline phosphatase, albumin, and creatine) will be measured by AniLytics Inc. (Gaithersburg, MD). Plasma insulin, leptin and IGF-1 levels will be determined by using an ELISA kit (ALPCO Diagnostics, NH).Glucose tolerance and insulin sensitivity. For glucose tolerance test, mice fasted for 8 h will be intraperitoneally (i.p.) injected with a dose of glucose (1 µg/kg body weight). For insulin sensitivity assays, non-fasting mice will be i.p. injected with insulin (Sigma-Aldrich, St. Louis, MO, 0.25 U/kg body weight). Immediately before and 0.5-2.5 h after the injections, glucose and insulin levels will be measured as described in C.1.Immunohistochemistry, Western analysis and molecular markers of adipogenesis. Frozen tissue samples will be sectioned and prepared by Histoserv, Inc. (Germantwon, MD). For immunofluorescence staining of insulin and glucagon in the pancreas and PPARγ and C/EBPα in adipocytes, the slides will be fixed in 4% PFA, permeabilized in 0.2% Triton X-100 in PBS, and blocked in 5% Normal Donkey Serum, 1% Bovine Serum Albumin, 0.2% GELATIN, 0.2% TRITON X-100 in PBS. Then, the tissue sections will be hybridized with insulin and glucagon antibody (1:500, Abcam, Cambridge, UK). The slides will be washed two times in 1X PBS with 0.2% Triton X-100, for 30 minutes each time and washed once in 1X PBS for 30 minutes. The washed slides will be hybridized with secondary antibody (in 1:200 dilution, anti-mouse Alexa fluor 488 for glucagon and anti-rabbit Alexa fluor 568 for insulin, Invitrogen, Carlsbad, CA), washed, and mounted by ProLong Gold Antifade Mountant with DAPI (Invitrogen, Carlsbad, CA). The above proteins, in addition to insulin-receptor β (IRβ), activated IRβ (p-IRβ), Akt, and activated Akt (P-Akt on Thr-308 and Ser-473, Cell signaling Inc.) in the liver, will be determined by Western analysis. For qPCR, organs will be stored in Trizol prior to RNA extraction by using chloroform and isopropanol (Fisher Scientific, Fair Lawn, New Jersey), and the extracted RNA will be subjected to reverse transcription by High-Capacity cDNA Reverse Transcription Kit (Invitrogen, Carlsbad, CA) following manufacturer's manual. Specific primers for insulin, glucagon, PPARα, and C/EBPα will be synthesized by Integrated DNA Technology (Coralville, Iowa). Primers will be proportionally mixed with Universal SYBR Green Supermix (Bio-rad, Hercules, California) and cDNA for real-time PCR. The mixtures will be loaded on ABI 7500 Fast Real-Time PCR System (Life Technology, Carlsbad, California) for data collection.